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Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31).

Litwin M, Clark K, Noack L, Furze J, Berndt M, Albelda S, Vadas M, Gamble J - J. Cell Biol. (1997)

Bottom Line: In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating.In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression.In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Immunology, Hanson Centre for Cancer Research, Adelaide, South Australia.

ABSTRACT
Tumor necrosis factor-alpha, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell-cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. In contrast, ECs failed to express E-selectin when plated on poly-L-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 +/- 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule-1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co- culture of subconfluent ECs with PECAM-1- coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.

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Adhesion through PECAM-1–coated beads downregulates E-selectin. ECs at subconfluent density (0.25 × 105  cells per cm2) were plated in the  absence or presence of beads  (20 beads per cell) coated either  with purified PECAM-1 or the  blocking agent (BSA) alone.  Cells at high density (105 cells  per cm2) were also plated. E-selectin expression was measured 18 h  after plating. The data represents three experiments where each  group was performed in triplicate and is presented relative to the  E-selectin expression on subconfluent ECs, which is shown as  100%. (The mean MFI at 100% was 2.01 ± 0.09.) Asterisk indicates where PECAM-1–coated beads significantly decreased  E-selectin (P < 0.001 by ANOVA) compared to BSA-coated  beads.
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Figure 9: Adhesion through PECAM-1–coated beads downregulates E-selectin. ECs at subconfluent density (0.25 × 105 cells per cm2) were plated in the absence or presence of beads (20 beads per cell) coated either with purified PECAM-1 or the blocking agent (BSA) alone. Cells at high density (105 cells per cm2) were also plated. E-selectin expression was measured 18 h after plating. The data represents three experiments where each group was performed in triplicate and is presented relative to the E-selectin expression on subconfluent ECs, which is shown as 100%. (The mean MFI at 100% was 2.01 ± 0.09.) Asterisk indicates where PECAM-1–coated beads significantly decreased E-selectin (P < 0.001 by ANOVA) compared to BSA-coated beads.

Mentions: The second approach used platelet purified PECAM-1 immobilized on beads. E-selectin expression on EC plated at low density in the presence of PECAM-1–coated beads was inhibited when measured 18 h after plating (inhibition was 61 ± 20%; n = 3). BSA-coated beads had no effect (Fig. 9). Interestingly, neither purified, soluble PECAM-1, nor its immobilization on plastic was able to regulate E-selectin expression (data not shown) suggesting that valency, concentration, or microenvironment problems may be operating.


Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31).

Litwin M, Clark K, Noack L, Furze J, Berndt M, Albelda S, Vadas M, Gamble J - J. Cell Biol. (1997)

Adhesion through PECAM-1–coated beads downregulates E-selectin. ECs at subconfluent density (0.25 × 105  cells per cm2) were plated in the  absence or presence of beads  (20 beads per cell) coated either  with purified PECAM-1 or the  blocking agent (BSA) alone.  Cells at high density (105 cells  per cm2) were also plated. E-selectin expression was measured 18 h  after plating. The data represents three experiments where each  group was performed in triplicate and is presented relative to the  E-selectin expression on subconfluent ECs, which is shown as  100%. (The mean MFI at 100% was 2.01 ± 0.09.) Asterisk indicates where PECAM-1–coated beads significantly decreased  E-selectin (P < 0.001 by ANOVA) compared to BSA-coated  beads.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139821&req=5

Figure 9: Adhesion through PECAM-1–coated beads downregulates E-selectin. ECs at subconfluent density (0.25 × 105 cells per cm2) were plated in the absence or presence of beads (20 beads per cell) coated either with purified PECAM-1 or the blocking agent (BSA) alone. Cells at high density (105 cells per cm2) were also plated. E-selectin expression was measured 18 h after plating. The data represents three experiments where each group was performed in triplicate and is presented relative to the E-selectin expression on subconfluent ECs, which is shown as 100%. (The mean MFI at 100% was 2.01 ± 0.09.) Asterisk indicates where PECAM-1–coated beads significantly decreased E-selectin (P < 0.001 by ANOVA) compared to BSA-coated beads.
Mentions: The second approach used platelet purified PECAM-1 immobilized on beads. E-selectin expression on EC plated at low density in the presence of PECAM-1–coated beads was inhibited when measured 18 h after plating (inhibition was 61 ± 20%; n = 3). BSA-coated beads had no effect (Fig. 9). Interestingly, neither purified, soluble PECAM-1, nor its immobilization on plastic was able to regulate E-selectin expression (data not shown) suggesting that valency, concentration, or microenvironment problems may be operating.

Bottom Line: In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating.In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression.In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Immunology, Hanson Centre for Cancer Research, Adelaide, South Australia.

ABSTRACT
Tumor necrosis factor-alpha, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell-cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. In contrast, ECs failed to express E-selectin when plated on poly-L-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 +/- 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule-1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co- culture of subconfluent ECs with PECAM-1- coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.

Show MeSH
Related in: MedlinePlus